Polymers with bioactive agents

ABSTRACT

The invention relates to a process for loading a polymer with one or more bioactive agents, using a wet spinning technique. The invention further relates to a polymer loaded with one or more bioactive agents, obtainable by said process and to the use thereof as a carrier for controlled drug release or as scaffold for tissue engineering.

[0001] The invention relates to a fibrous polymer loaded with one ormore bioactive agents and to a process for preparing the fibrous polymerloaded with the bioactive agent or agents. The invention further relatesto the use of the polymer loaded with the bioactive agent or agents as ascaffold for tissue engineering.

[0002] The development of biological substitutes, that can restore orimprove tissue function, is a rapidly evolving interdisciplinary fieldin science. New tissues can be engineered from living cells and threedimensional scaffolds. The function of the scaffold is to providestructural integrity and space for growing tissue, and to guide tissueformation. For this purpose, scaffolds are needed with a high porosityand a high surface area. Ideally, the scaffold delivers bioactivefactors which modulate cellular behavior such as proliferation,migration and adhesion. For example, it has been shown that release ofbone morphogenetic protein (rhBMP-2) from biodegradable porous scaffoldsstimulated growth of bone into the scaffolds in vivo (see K. Whang etal., J. Biomed. Mater. Res. 42 (1998) 491-499).

[0003] Macroporous scaffolds for tissue engineering have been fabricatedby various techniques, including fiber bonding (see A. G. Mikos et al.,J. Biomed. Mater. Res. 27 (1993) 183-189), solvent casting/salt-leaching(see A. G. Mikos et al., Biomaterials 14 (1993) 323-330), phaseseparation (see H. Lo et al., J. Biomed. Mater. Res. 30 (1996) 475-484)and emulsion freeze-drying (see K. Whang et al., Polymer 36 (1995)837-842). Often, the methods used to prepare macroporous structures arenot suitable for incorporation of labile proteins and other bioactivecompounds, due to the high temperatures used, exposure to organicsolvents, or the need for removal of the porogens.

[0004] Recently, Whang et al. (see J. Biomed. Mater. Res. 42 (1998)491-499) developed an emulsion freeze-drying process to overcome thesedrawbacks in the incorporation of proteins into porous matrices. Thismethod consists of creating an emulsion from apoly(lactide-co-glycolide) (PLG) solution in methylene chloride and anaqueous protein solution. Subsequently, the emulsion is quenched inliquid nitrogen, and methylene chloride and water are removed byfreeze-drying. The large pores in the resulting matrices are formed bythe dispersed water phase and since the proteins are also dissolved inthe water phase, this implies that the proteins are located within thelarge interconnected pores. This might limit the possibilities to obtainslow release of proteins. Furthermore, it appeared that the type ofprotein influenced the ultimate structure of the pores. In case ofbovine serum albumin (BSA) loaded scaffolds, the median pore size was 65μm, while incorporation of rhBMP-2 resulted in a median pore size ofonly 9 μm, which is probably too small for optimal bone-ingrowth.

[0005] The present invention aims to provide a method for preparing afibrous polymer loaded with one or more bioactive agents. Further, inparticular in view of the application of polymers as scaffold for tissueengineering, it is often desired to be able to incorporate (bioactive)additives in a solid body that constitutes the scaffold. For instance,the presence of growth factors may be very much desired in order toenhance cell growth or differentiation. As many of these bioactiveadditives are very sensitive compounds, the need for working under mildconditions becomes even more important. It is particularly desired thatthe method can be performed under such mild conditions that the biologicactivity of the bioactive agent is essentially not deteriorated duringthe carrying out of the method. Further, it is desired that thebioactive agent can be homogeneously distributed throughout the polymer.

[0006] Surprisingly, it has now been found that a wet spinning techniqueis highly suitable for achieving the above goals. Accordingly, theinvention specifically relates to a process for preparing a polymerloaded with one or more bioactive agents comprising the steps of:

[0007] a) providing a solution of the polymer in a suitable firstsolvent;

[0008] b) adding an aqueous solution of the bioactive agent or agents tothe polymer solution to obtain a water-in-oil emulsion;

[0009] c) immersing the water-in-oil emulsion in a suitable secondsolvent by injecting the emulsion through a nozzle into the secondsolvent;

[0010] d) allowing the first solvent to migrate into the second solventto obtain a solid, fibrous polymer loaded with the bioactive agent oragents.

[0011] The present process is carried out under mild conditions; no hightemperatures or extreme pH is required. As a result, the stability andactivity of the bioactive agent or agents is essentially maintainedduring the process. Furthermore, it has been found possible in a processaccording to the invention to obtain a polymeric substrate in which thebioactive agent is homogeneously distributed. Another advantage is thatthe present process yields a fibrous product, which is believed to be ahighly suitable form for scaffolds in tissue engineering, enablingdiffusion of nutrients and waste materials to and from cells seeded onthe scaffold and mimicking natural fibrous tissues, such as muscletissue. Furthermore the present product may find advantageousapplication in the field of surgical devices and aids, for instance asdevice for controlled release of bioactive agents in vivo. Specificexamples of such devices are spacers that may be used to release anantibiotic, such as gentamycin, in case of an infection, for examplewhen a revision hip implant is to be inserted in a patient, or devicesfor the release of anti-conception agents.

[0012] The polymer which is loaded according to the present inventionmay be any kind of polymer. Preferably, the polymer is a biocompatiblepolymer, thus enabling the use of the polymer, loaded with the bioactiveagent, for pharmaceutical and/or biological purposes. In the context ofthe present invention, the term biocompatible is intended to refer tomaterials which may be incorporated into a human or animal bodysubstantially without unacceptable responses of the human or animal. Itis further preferred that the polymer is a biodegradable polymer, whichmakes the polymer loaded with bioactive agent(s) highly suitable for useas a scaffold in tissue engineering. The term biodegradable refers tomaterials which, after a certain period of time, are broken down in abiological environment. Preferably, the rate of breakdown is chosensimilar or identical to the rate at which the body generates autogenoustissue to replace an implant manufactured of the biodegradable material.

[0013] Suitable examples of polymers to be loaded with one or morebioactive agents in accordance with the invention are amphiphilic blockcopolymers, comprising hydrophilic and hydrophobic blocks. Thehydrophilic component is preferably a polyalkylene glycol, such aspolyethylene glycol. The hydrophobic blocks may be chosen from a varietyof possibilities, including poly(lactide-co-glycolide),poly(caprolactone), polybutylene terephtalate, poly(propylene fumarate),and poly(anhydrides). Such block copolymers may be diblock, triblock,multiblock or star-shaped block copolymers. It has been found that theuse of these polymers lead to very stable emulsions, which beneficiallyaffects the formation of the polymer fibers.

[0014] A preferred class of polymers according to the invention, is acopolymer of a polyalkylene glycol terephtalate and an aromaticpolyester. Preferably, the copolymer comprises 20-90 wt. %, morepreferably 40-70 wt. % of the polyalkylene glycol terephtalate, and80-10 wt. %, more preferably 60-30 wt. % of the aromatic polyester. Apreferred type of copolymers according to the invention is formed by thegroup of block copolymers.

[0015] The polyalkylene glycol terephtalate may have a weight averagemolecular weight of about 150 to about 4000. Preferably, thepolyalkylene glycol terephtalate has a weight average molecular weightof 200 to 1500. The aromatic polyester preferably has a weight averagemolecular weight of from 200 to 5000, more preferably from 250 to 4000.The weight average molecular weight of the copolymer preferably liesbetween 10,000 and 300,000, more preferably between 40,000 and 120,000.

[0016] The weight average molecular weight may suitably be determined bygel permeation chromatography (GPC). This technique, which is known perse, may for instance be performed using chloroform as a solvent andpolystyrene as external standard. Alternatively, a measure for theweight average molecular weight may be obtained by using viscometry (seeNEN-EN-ISO 1628-1). This technique may for instance be performed at 25°C. using chloroform as a solvent. Preferably, the intrinsic viscosity ofthe copolymer lies between 0.2289 and 1.3282 dL/g, which corresponds toa weight average molecular weight between 10,000 and 200,000. Likewise,the more preferred ranges for the weight average molecular weightmeasured by GPC mentioned above can also be expressed in terms of theintrinsic viscosity.

[0017] In a preferred embodiment, the polyalkylene glycol terephtalatecomponent has units of the formula —OLO—CO-Q-CO—, wherein O representsoxygen, C represents carbon, L is a divalent organic radical remainingafter removal of terminal hydroxyl groups from apoly(oxyalkylene)glycol, and Q is a divalent organic radical.

[0018] Preferred polyalkylene glycol terephtalates are chosen from thegroup of polyethylene glycol terephtalate, polypropylene glycolterephtalate, and polybutylene glycol terephtalate and copolymersthereof, such as poloxamers. A highly preferred polyalkylene glycolterephtalate is polyethylene glycol terephtalate.

[0019] The terms alkylene and polyalkylene generally refer to anyisomeric structure, i.e. propylene comprises both 1,2-propylene and1,3-propylene, butylene comprises 1,2-butylene, 1,3-butylene,2,3-butylene, 1,2-isobutylene, 1,3-isobutylene and 1,4-isobutylene(tetramethylene) and similarly for higher alkylene homologues. Thepolyalkylene glycol terephtalate component is preferably terminated witha dicarboxylic acid residue —CO-Q-CO—, if necessary to provide acoupling to the polyester component. Group Q may be an aromatic grouphaving the same definition as R, or may be an aliphatic group such asethylene, propylene, butylene and the like.

[0020] The polyester component preferably has units —O-E-O—CO—R—CO—,wherein O represents oxygen, C represents carbon, E is a substituted orunsubstituted alkylene or oxydialkylene radical having from 2 to 8carbon atoms, and R is a substituted or unsubstituted divalent aromaticradical.

[0021] In a preferred embodiment, the polyester is chosen from the groupof polyethylene terephthalate, polypropylene terephthalate, andpolybutylene terephthalate. A highly preferred polyester is polybutyleneterephthalate.

[0022] The preparation of the copolymer will now be explained by way ofexample for a polyethylene glycol terephtalate/polybutyleneterephthalate copolymer. Based on this description, the skilled personwill be able to prepare any desired copolymer within the above describedclass. An alternative manner for preparing polyalkylene glycolterephtalate/polyester copolymers is disclosed in U.S. Pat. No.3,908,201.

[0023] A polyethylene glycol terephtalate/polybutylene terephthalatecopolymer may be synthesized from a mixture of dimethyl terephthalate,butanediol (in excess), polyethylene glycol, an antioxidant and acatalyst. The mixture is placed in a reaction vessel and heated to about180° C., and methanol is distilled as transesterification proceeds.During the transesterification, the ester bond with methyl is replacedwith an ester bond with butylene and/or the polyethyene glycol. Aftertransesterification, the temperature is raised slowly to about 245° C.,and a vacuum (finally less than 0.1 mbar) is achieved. The excessbutanediol is distilled off and a prepolymer of butanediol terephthalatecondenses with the polyethylene glycol to form apolyethylene/polybutylene terephthalate copolymer. A terephthalatemoiety connects the polyethylene glycol units to the polybutyleneterephthalate units of the copolymer and thus such a copolymer also issometimes referred to as a polyethylene glycolterephthalate/polybutylene terephthalate copolymer (PEGT/PBT copolymer).

[0024] The bioactive agent which is to be loaded into the polymer may bechosen from various groups of compounds. The term “biologically activeagent” or bioactive agent, as used herein, includes an agent whichprovides a therapeutic or prophylactic effect, a compound that affectsor participates in tissue growth, cell growth, cell differentiation, acompound that may be able to invoke a biological action such as animmune response, or could play any other role in one or more biologicalprocesses. Such agents include, but are not limited to, antimicrobialagents (including antibacterial and anti-fungal agents), anti-viralagents, anti-tumor agents, hormones, immunogenic agents, growth factors,lipids, lipopolysaccharides, and peptides, polypeptides and proteins ingeneral.

[0025] An important group of compounds that can be used for loading apolymer according to the invention is formed by peptides and proteins,of which in principle any kind may be incorporated according to thepresent invention. Both peptides and proteins are compounds that arebuilt up out of amino acids, linked to one another via an amide bond (orpeptide bond). This bond is the product of the joining of an amino groupof one amino acid with a carboxylic acid group of the other. Relativelysmall peptides may be referred to by the number of amino acids (e.g.di-, tri-, tetrapeptides). A peptide with a relatively small number ofamide bonds may also be called an oligopeptide, whereas a peptide with arelatively high number may be called a polypeptide or protein. Inaddition to being a polymer of amino acid residues, certain proteins mayfurther be characterized by the so called quaternary structure, aconglomerate of a number of polypeptides that are not necessarilychemically linked by amide bonds but are bonded by forces generallyknown to the skilled professional, such as electrostatic forces andvanderwaals forces. The term peptides, proteins or mixtures thereof asused herein is to include all above mentioned possibilities.

[0026] Usually, the protein and/or peptide will be selected on the basisof its biological activity. Depending on the type of polymer chosen, theproduct obtainable by the present process is highly suitable forcontrolled release of proteins and peptides. In a preferred embodiment,the protein or peptide is a growth factor. A growth factor is defined asa protein or peptide that has a beneficial effect on the growth,proliferation and/or differentiation of living cells. According to thisembodiment, the process of the invention provides a material that canadvantageously be used as a scaffold for tissue engineering, wherein thegrowth factor is released from the polymer in a delayed manner, thusproviding a beneficial environment for tissue to grow and/ordifferentiate.

[0027] Examples of preferred growth factors are Bone MorphogeneticProteins (BMP), epidermal growth factors, e.g. Epidermal Growth Factor(EGF), fibroblast growth factors, e.g. basic Fibroblast Growth Factor(bFGF), Nerve Growth Factor (NGF), Bone Derived Growth Factor (BDGF),transforming growth factors, e.g. Transforming Growth Factor-β1(TGF-β1), and human Growth Hormone (hGH).

[0028] Further examples of peptides or proteins or entities comprisingpeptides or proteins which may advantageously be contained in the loadedpolymer include, but are not limited to, immunogenic peptides orimmunogenic proteins, which include, but are not limited to, thefollowing:

[0029] 1. Toxins: diphtheria toxin, tetanus toxin

[0030] 2. Viral surface antigens or parts of viruses: adenoviruses,Epstein-Barr Virus, Hepatitis A Virus, Hepatitis B Virus, Herpesviruses, HIV-1, HIV-2, HTLV-III, Influenza viruses, Japaneseencephalitis virus, Measles virus, Papilloma viruses, Paramyxoviruses,Polio Virus, Rabies, Virus, Rubella Virus, Vaccinia (Smallpox) viruses,Yellow Fever Virus

[0031] 3. Bacterial surface antigens or parts of bacteria: Bordetellapertussis, Helicobacter pylori, Clostridium tetani, Corynebacteriumdiphtheria, Escherichia coli, Haemophilus influenza, Klebsiella species,Legionella pneumophila, Mycobacterium bovis, Mycobacterium leprae,Mycrobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Proteus species, Pseudomonas aeruginosa, Salmonellaspecies, Shigella species, Staphylococcus aureus, Streptococcuspyogenes, Vibrio cholera, Yersinia pestis

[0032] 4. Surface antigens of parasites causing disease or portions ofparasites: Plasmodium vivax—malaria, Plasmodium falciparum—malaria,Plasmodium ovale—malaria, Plasmodium malariae—malaria, Leishmaniatropica—leishmaniasis, Leishmania donovani, leishmaniasis, Leishmaniabranziliensis—leishmaniasis, Trypanosoma rhodescense—sleeping sickness,Trypanosoma gambiense—sleeping sickness, Trypanosoma cruzi—Chagas'disease, Schistosoma mansoni—schistosomiasis, Schistosomomahaematobium—schistomiasis, Schistosoma japonicum—shichtomiasis,Trichinella spiralis—trichinosis, Stronglyloides duodenale—hookworm,Ancyclostoma duodenale—hookworm, Necator americanus—hookworm, Wucheriabancrofti—filariasis, Brugia malaya—filariasis, Loa loa—filariasis,Dipetalonema perstaris—filariasis, Dracuncula medinensis—filariasis,Onchocerca volvulus—filariasis

[0033] 5. Immunoglobulins: IgG, IgA, IgM, Antirabies immunoglobulin,Antivaccinia immunoglobulin

[0034] 6. Anititoxins: Botulinum antitoxin, diphtheria antitoxin, gasgangrene antitoxin, tetanus antitoxin.

[0035] 7. Antigens which elicit an immune response against: Foot andMouth Disease, hormones and growth factors such as follicle stimulatinghormone, prolactin, angiogenin, epidermal growth factor, calcitonin,erythropoietin, thyrotropic releasing hormone, insulin, growth hormones,insulin-like growth factors 1 and 2, skeletal growth factor, humanchorionic gonadotropin, luteinizing hormone, nerve growth factor,adrenocorticotropic hormone (ACTH), luteinizing hormone releasinghormone (LHRH), parathyroid hormone (PTH), thyrotropin releasing hormone(TRH), vasopressin, cholecystokinin, and corticotropin releasinghormone; cytokines, such as interferons, interleukins, colonystimulating factors, and tumor necrosis factors: fibrinolytic enzymes,such as urokinase, kidney plasminogen activator; and clotting factors,such as Protein C, Factor VIII, Factor IX, Factor VII and AntithrombinIII.

[0036] 8. Examples of other proteins or peptides: albumin, atrialnatriuretic factor, renin, superoxide dismutase, α₁-antitrypsin, lungsurfactant proteins, bacitracin, bestatin, cydosporine, deltasleep-inducing peptide (DSIP), endorphins, glucagon, gramicidin,melanocyte inhibiting factors, neurotensin, oxytocin, somostatin,terprotide, serum thymide factor, thymosin, DDAVP, dermorphin,Met-enkephalin, peptidoglycan, satietin, thymopentin, fibrin degradationproduct, des-enkephalin-α-endorphin, gonadotropin releasing hormone,leuprolide, α-MSH, and metkephamid.

[0037] It is to be understood, however, that the scope of the presentinvention is not limited to any specific peptides or proteins.

[0038] Although, in view of the delicacy of proteins and peptides, thepresent process is particularly useful for making polymers loaded withproteins and peptides, it is of course also possible to load a polymerwith a substance other than a protein or peptide. Such biologicallyactive agents which may be incorporated include, but are not limited to,non-peptide, non-protein drugs. It is possible within the scope of thepresent invention to incorporate drugs of a polymeric nature, but alsoto incorporate drugs of a relatively small molecular weight of less than1500, or even less than 500.

[0039] Examples of non-peptide, non-protein drugs which may beincorporated include, but are not limited to, the following:

[0040] 1. Anti-tumor agents: altretamin, fluorouracil, amsacrin,hydroxycarbamide, asparaginase, ifosfamid, bleomycin, lomustin,busulfan, melphalan, chlorambucil, mercaptopurin, chlormethin,methotrexate, cisplatin, mitomycin, cyclophosphamide, procarbazin,cytarabin, teniposid, dacarbazin, thiotepa, dactinomycin, tioguanin,daunorubicin, treosulphan, doxorubicin, tiophosphamide, estramucin,vinblastine, etoglucide, vincristine, etoposid, vindesin.

[0041] 2. Anitimicrobial agents

[0042] 2.1 Antibiotics

[0043] Penicillins: ampicillin, nafcillin, amoxicillin, oxacillin,azlocillin, penicillin G, carbenicillin, penicillin V, dicloxacillin,phenethicillin, floxacillin, piperacillin, mecillinam, sulbenicillin,methicillin, ticarcillin, mezlocillin

[0044] Cephalosporins: cefaclor, cephalothin, cefadroxil, cephapirin,cefamandole, cephradine, cefatrizine, cefsulodine, cefazolin,ceftazidim, ceforanide, ceftriaxon, cefoxitin, cefuroxime, cephacetrile,latamoxef, cephalexin

[0045] Aminoglycosides: amikacin, neomycin, dibekacyn, kanamycin,gentamycin, netilmycin, kanamycin, tobramycin

[0046] Macrolides: amphotericin B, novobiocin, bacitracin, nystatin,clindamycin, polymyxins, colistin, rovamycin, erythromycin,spectinomycin, lincomycin, vancomycin

[0047] Tetracyclines: chlortetracycline, oxytetracycline,demeclocycline, rolitetracycline, doxycycline, tetracycline, minocyclineOther antibiotics: chloramphenicol, rifamycin, rifampicin, thiamphenicol

[0048] 2.2 Chemotherapeutic agents

[0049] Sulfonamides: sulfadiazine, sulfamethizol, sulfadimethoxin,sulfamethoxazole, sulfadimidin, sulfamethoxypyridazine, sulfafurazole,sulfaphenazol, sulfalene, sulfisomidin, sulfamerazine, sulfisoxazole,trimethoprim with sulfamethoxazole or sulfametrole

[0050] Urinary tract antiseptics: methanamine, quinolones(norfloxacin,cinoxacin), nalidixic acid, nitro-compounds (nitrofurantoine,nifurtoinol), oxolinic acid

[0051] Anaerobic infections: metronidazole

[0052] 3. Drugs for tuberculosis: aminosalicyclic acid, isoniazide,cycloserine, rifampicine, ethambutol, tiocarlide, ethionamide, viomycin

[0053] 4. Drugs for leprosy: amithiozone, rifampicine, clofazimine,sodium sulfoxone, diaminodiphenylsulfone (DDS, dapsone)

[0054] 5. Antifungal agents: amphotericin B, ketoconazole, clotrimazole,miconazole, econazole, natamycin, flucytosine, nystatine, griseofulvin

[0055] 6. Antiviral agents: aciclovir, idoxuridine, amantidine,methisazone, cytarabine, vidarabine, ganciclovir

[0056] 7. Chemotherapy of amebiasis: chloroquine, iodoquinol,clioquinol, metronidazole, dehydroemetine, paromomycin, diloxanide,furoatetinidazole, emetine

[0057] 8. Anti-malarial agents: chloroquine, pyrimethamine,hydroxychloroquine, quinine, mefloquine, sulfadoxine/pyrimethamine,pentamidine, sodium suramin, primaquine, trimethoprim, proguanil

[0058] b 9. Anti-helminthiasis agents: antimony potassium tartrate,niridazole, antimony sodium dimercaptosuccinate, oxamniquine, bephenium,piperazine, dichlorophen, praziquantel, diethylcarbamazine, pyrantelparmoate, hycanthone, pyrivium pamoate, levamisole, stibophen,mebendazole, tetramisole, metrifonate, thiobendazole, niclosamide

[0059] 10. Anti-inflammatory agents: acetylsalicyclic acid, mefenamicacid, aclofenac, naproxen, azopropanone, niflumic acid, benzydamine,oxyphenbutazone, diclofenac, piroxicam, fenoprofen, pirprofen,flurbiprofen, sodium salicyclate, ibuprofensulindac, indomethacin,tiaprofenic acid, ketoprofen, tolmetin

[0060] 11. Anti-gout agents: colchicine, allopurinol

[0061] 12. Centrally acting (opoid) analgesics: alfentanil, methadone,bezitramide, morphine, buprenorfine, nicomorphine, butorfanol,pentazocine, codeine, pethidine, dextromoramide, piritranide,dextropropoxyphene, sufentanil, fentanyl

[0062] 13. Local anesthetics: articaine, mepivacaine, bupivacaine,prilocaine, etidocaine, procaine, lidocaine, tetracaine

[0063] 14. Drugs for Parkinson's disease: amantidine, diphenhydramine,apomorphine, ethopropazine, benztropine mesylate, lergotril, biperiden,levodopa, bromocriptine, lisuride, carbidopa, metixen, chlorphenoxamine,orphenadrine, cycrimine, procyclidine, dexetimide, trihexyphenidyl

[0064] 15. Centrally active muscle relaxants: baclofen, carisoprodol,chlormezanone, chlorzoxazone, cyclobenzaprine, dantrolene, diazepam,febarbamate, mefenoxalone, mephenesin, metoxalone, methocarbamol,tolperisone

[0065] 16. Hormones and hormone antago7nistics

[0066] 16.1 Corticosteroids

[0067] 16.1.1 Mineralocorticosteroids: cortisol, desoxycorticosterone,flurohydrocortisone

[0068] 16.1.2 Glucocorticosteroids: beclomethasone, betamethasone,cortisone, dexamethasone, fluocinolone, fluocinonide, fluocortolone,fluorometholone, fluprednisolone, flurandrenolide, halcinonide,hydrocortisone, medrysone, methylprednisolone, paramethasone,prednisolone, prednisone, triamcinolone (acetonide)

[0069] 16.2 Androgents

[0070] 16.2.1 Androgenic steroids used in therapy: danazole,fluoxymesterone, mesterolone, methyltestosterone, testosterone and saltsthereof

[0071] 16.2.2 Anabolic steroids used in therapy: calusterone, nandroloneand salts thereof, dromostanolone, oxandrolone, ethylestrenol,oxymetholone, methandriol, stanozolol methandrostenolone, testolactone

[0072] 16.2.3 Antiandrogens: cyproterone acetate

[0073] 16.3 Estrogens

[0074] 16.3.1 Estrogenic steroids used it therapy: diethylstilbestrol,estradiol, estriol, ethinylestradiol, mestranol, quinestrol

[0075] 16.3.2 Anti-estrogens: chlorotrianisene, clomiphene,ethamoxytriphetol, nafoxidine, tamoxifen

[0076] 16.4 Progestins: allylestrenol, desogestrel, dimethisterone,dydrogesterone, ethinylestrenol, ethisterone, ethynadiol diacetate,etynodiol, hydroxyprogesterone, levonorgestrel, lynestrenol,medroxyprogesterone, megestrol acetate, norethindrone, norethisterone,norethynodrel, norgestrel, progesterone

[0077] 17. Thyroid drugs

[0078] 17.1 Thyroid drugs used in therapy: levothyronine, liothyronine

[0079] 17.2 Anti-thyroid drugs used in therapy: carbimazole,methimazole, methylthiouracil, propylthiouracil

[0080] When a hydrophobic drug, such as, for example, a steroid hormoneis incorporated, preferably at least one hydrophobic antioxidant ispresent. Hydrophobic antioxidants which may be employed include, but arenot limited to, tocopherols, such as α-tocopherol, β-tocopherol,γ-tocopherol, δ-tocopherol, ε-tocopherol, ζ₁-tocopherol, ζ₂ -tocopherol,and η-tocopherol; and 1-ascorbic acid 6-palmitate. Such hydrophobicantioxidants retard the degradation of the copolymer and retard therelease of the biologically active agent. Thus, the use of a hydrophobicor lipophilic antioxidant is applicable particularly to the formation ofloaded polymers which include drugs which tend to be released quickly,such as, for example, drug molecules having a molecular weight less than500. The hydrophobic antioxidant(s) may be present in the loaded polymerin an amount of from about 0.1 wt. % to about 10 wt. % of the totalweight of the polymer, preferably from about 0.5 wt. % to about 2 wt. %.

[0081] When the loaded polymer includes a hydrophilic drug, such as anaminoglycoside, the loaded polymer may also include, in addition to ahydrophobic antioxidant, a hydrophobic molecule such as cholesterol,ergosterol, lithocholic acid, cholic acid, dinosterol, betuline, oroleanolic acid, which may be employed in order to retard the releaserate of the agent from the copolymer. Such hydrophobic molecules preventwater penetration into the loaded polymer, but do not compromise thedegradability of the polymer matrix. In addition, such molecules havemelting points from 150° C. to 200° C. or decreases the polymer matrixdiffusion coefficient for the biologically active agent, such as drugmolecule, to be released. Thus, such hydrophobic molecules provide for amore sustained release of a biologically active agent from the polymermatrix. The at least one hydrophobic molecule may be present in theloaded polymer in an amount of from about 0.1 wt. % to about 20 wt. %,preferably from 1.0 wt. % to 5.0 wt. %.

[0082] It is noted that, for the preparation of the water-in-oilemulsion according to the invention, it is necessary that a hydrophobicbioactive agent dissolves at least slightly in water, preferably atleast to such an extent that the resultant loaded polymer comprises anamount of the bioactive agent which is sufficient to achieve a desiredeffect in vivo. If necessary, a surfactant may be added to the aqueoussolution of the bioactive agent in order to achieve that a minimaldesired amount of the bioactive agent is incorporated into the polymer.Examples of such surfactants are well known to the skilled artisan andmay be used in amounts which can easily be optimized by the artisanbased on his normal knowledge of the art. Specific examples of suitablesurfactants include, but are not limited to, poly(vinyl)alcohol, Span80, Tween and Pluronics.

[0083] The invention further requires the use of two solvents which arechosen to complement each other's action in the present process. Thefirst solvent is to be chosen such that it is immiscible with water. Inaddition, the polymer which is to be loaded with bioactive agent(s)should be soluble in the first solvent. The second solvent is to bechosen such that the polymer is not soluble in it. Also, the firstsolvent is to be well miscible with the second solvent. Preferably, thefirst solvent mixes better with the second solvent than that the polymerdissolves in the first solvent. This ensures that, upon immersion of thewater-in-oil emulsion in the second solvent, the first solvent willsubstantially completely migrate into the second solvent. Furtherpreferred is that both solvents are immiscible with water. This makes itpossible to prevent that the bioactive agent, which is processed in anaqueous solution, comes into contact with an organic solvent, whichmight be harmful to bioactive agent. Depending on the nature of thepolymer to be loaded, the skilled person will be able to select suitablesolvents. By way of example, good results have been obtained by usingchloroform as the first solvent, and hexane as the second solvent whenthe polymer is polyethylene glycol terephtalate/polybutyleneterephthalate copolymer.

[0084] In a first step of the present process, a solution is provided ofthe polymer in the first solvent. The concentration of this solution isnot critical. On the one hand, it is important that all of the polymerdissolves. On the other hand, it is preferred that the amount of thefirst solvent used is kept as small as possible in order to keep theprocess efficient.

[0085] Of the polymer solution, a water-in-oil emulsion is prepared bymixing it with an aqueous solution of the bioactive agent(s),. Undercertain circumstances, it may be desired to add conventional stabilizersfor enhancing the stability of the water-in-oil emulsion. Typicalexamples of such stabilizers include proteins such as albumin or casein,Pluronics and Span 80. It is, however, preferred that such stabilizersare not used.

[0086] The amount of bioactive agent(s), in the aqueous solution will bechosen such that a desired amount of these bioactive agents iseventually incorporated into the polymer. Depending on the type ofpolymer and the nature of the bioactive agent(s), the amount ofincorporated agent may vary. For proteins and peptides, for example, ithas been found that various proteins and peptides can be incorporatedinto the polymer in concentrations up to 10 wt. %, based on the weightof the loaded polymer. When using particularly hydrophilic bioactiveagents, such as the protein leuprolide, it has even been found possibleto incorporate the agent into the polymer in a concentration of up to 50wt. %, based on the weight of the loaded polymer. The lower limit of theamount of bioactive agent(s), is not critical and will depend on theactivity of the bioactive agent(s), and on the envisaged application ofbioactive agent loaded polymer. In the case of proteins and peptidestypically, at least 0.01 wt. %, based on the weight of the loadedpolymer, of protein and/or peptide will be incorporated.

[0087] The amount of water used for preparing the aqueous bioactiveagent solution will be at least so high as to enable an efficientdissolution of the bioactive agent without employing unduly harshconditions that might adversely affect the stability and/or biologicalactivity of the bioactive agent. The upper limit of the amount of waterused will depend on the rate at which the bioactive agent is to bereleased from the polymer in a final, envisaged application of thebioactive agent loaded polymer. It has been found that the use of largeramounts of water, leads to higher release rates of the polymer.Typically, the aqueous solution of the bioactive agent(s) will comprisebetween 0.001 and 10 wt. % of bioactive agent(s), based on the weight ofthe solution. In practice, the amount of bioactive agents in thesolution will depend on the solubility of the bioactive agents and onthe stability of the water-in-oil emulsion.

[0088] The obtained water-in-oil emulsion is next immersed in the secondsolvent by injection through a nozzle. The diameter and shape of thenozzle can be varied to obtain fibers of different thickness and shape.The injection itself will usually be driven by a pressure by virtue ofwhich the emulsion is transported through the nozzle into the secondsolvent. The injection may for instance be accomplished by use of asyringe or an extruder. The amount of the second solvent is notcritical. It should be at least sufficient for the emulsion to becompletely immersed in it and to allow a substantially completemigration of the first solvent from the emulsion into the secondsolvent. The upper limit will generally chosen on the basis of economicconsiderations.

[0089] Upon immersion of the emulsion into the second solvent, due tothe specific selection of the first and second solvents, the firstsolvent will migrate from the emulsion into the second solvent. Inpractice, it may often be observed that first exchange of the first andsecond solvents takes place, before the first solvent will migrate intothe second solvent. This may have the effect that the polymer fibers areprovided with a porosity. This phenomenon and how it may be controlledto obtain a desired porosity has been described by P. van de Witte,“Polylactide membranes. Correlation between phase transitions andmorphology”, PhD thesis, University of Twente, Enschede, 1994.

[0090] As a result, the polymer, which does not dissolve in the secondsolvent, will solidify thereby incorporating the bioactive agent(s).Finally, the solid loaded polymer may be removed from the mixture offirst and second solvents in any conventional manner and may eventuallybe dried.

[0091] In a preferred embodiment, the obtained fibers may be formed intoa fibrous mesh by collecting the fibers in a mold, and bonding themtogether by use of a suitable solvent mixture. This mixture shouldcomprise at least one solvent in which the polymer dissolves and atleast one solvent in which the polymer does not dissolve. Preferably, amixture is used of the above described first and second solvents. Thesecond solvent will generally be present in an amount exceeding that ofthe first solvent, in order to avoid the risk of any of the polymerdissolving in the solvent mixture. Preferably, the volumetric ratio ofthe first solvent to the second solvent lies between 1:1 and 1:3.

[0092] It will be understood that the invention also encompasses abioactive agent loaded polymer obtainable by the process as set forthherein above. Said polymer loaded with one or more bioactive agents maybe used in biological, pharmaceutical and surgical applications, whereina (controlled) release of a bioactive agent(s) from a polymericsubstrate is desired. Examples of such applications include, but are notlimited to, carriers for controlled drug release and scaffolds fortissue engineering.

[0093] The invention will now be elucidated by the following,non-restrictive examples.

EXAMPLES Materials and Methods Materials

[0094] Poly(ethylene glycol)terephthalate/poly(butylene terephthalate)multiblock copolymers (PEG/PBT) were obtained from IsoTis BV, Bilthoven,The Netherlands. The copolymers contained 30 wt % PBT and the PEGsegment length was 1000 g/mole (1000PEG70PBT30). Phosphate bufferedsaline (PBS), pH 7.4 was purchased from NPBI (Emmercompascuum, TheNetherlands). Bovine serum albumin (BSA, heat shock fractionate,fraction V powder minimum 98%) was purchased from Sigma Chem. Corp. (St.Louis, USA). All solvents used were of analytical grade.

Preparation of Bioactiue Agent Loaded PEG/PBT Fiber Meshes

[0095] The bioactive agent in this example was a protein. Protein loadedPEG/PBT fibers were prepared from water-in-oil emulsions. To producesuch emulsions, 3 or 3.5 ml of a protein solution in PBS (containing 25mg/ml BSA) was emulsified in a solution of 2 g PEG/PBT in 14 ml CHCl₃using ultra-turrax-mixing (30 s at 20.5 krpm, Ika Labortechnik T25).Subsequently, the emulsion was poured into a 20 ml glass syringe (BectonDickinson Multifit) equipped with a 0.4 mm needle (Neolus Terumo12G×1.5″). The emulsion was pushed through the needle into a beakercontaining 2 l hexane at a speed of 0.5 ml/min. by means of a perfusionpump (Secura E, B. Braun). The hexane bath was stirred at 300 rpm. toprevent premature sticking of the fibers. After fiber formation wascompleted, the fibers were collected and transferred to a glass mold ofthe desired shape (cylindrical, 5 cm diameter and 1.7 cm height). Tobond the fibers, a mixture of hexane and CHCl₃ (7:3, 3:2, or 1:1, v/v)was introduced into the mold. After drying overnight under atmosphericconditions, the fiber structures were freeze dried for 3 days, andstored at −40° C.

Scanning Electron Microscopy (SEM)

[0096] A Hitachi S-800 field emission SEM was used to evaluate thesurface characteristics and internal structure of fibrous scaffolds. Thedevices were cut in liquid nitrogen and mounted on a substrate holder.Samples were sputter-coated with a thin gold layer.

In vitro Release of Bioactive Agent

[0097] Protein loaded fiber meshes (approximately 40 mg) were incubatedin 5 ml PBS (pH 7.4). Vials were continuously shaken at 37° C. andsamples were taken at various time points. Protein content wasdetermined using a standard Coomassie Blue assay (Pierce). Buffer wasrefreshed after sampling.

Results and Discussion Matrix Characterization

[0098] Bonded fiber meshes, containing BSA, were prepared in athree-step procedure. First, a water-in-oil emulsion was formed from anaqueous protein solution and a polymer solution in CHCl₃. The secondstep involves wet spinning of the w/o emulsion into a hexane bath.Hexane is miscible with CHCl₃, but is a non-solvent for the PEG/PBTcopolymers. Consequently, extrusion of the fibers into hexane results insolidification of the fibers, due to exchange of solvent andnon-solvent. Since hexane is not miscible with water, contact betweenthe incorporated proteins and hexane is prevented as much as possible.

[0099]FIG. 1 shows scanning electron micrographs of a cross-section ofthe obtained protein loaded PEG/PBT fibers (magnification is 500× (A) or2000× (B)). The fiber cross-section was not circular (FIG. 1a). This isprobably caused by the shape of the needle. The surface of the fiberswas porous, whereas the interior of the fibers seemed to be dense. Thisis in contrast with the morphology of protein loaded PEG/PBT matrices,prepared by immersion precipitation of w/o emulsion droplets in hexane.These structures showed a porous internal morphology (data not shown).Probably, the morphology of the fibers was changed during the fiberbonding step in the solvent/non-solvent mixture. Furthermore, it cannotbe excluded that the internal structure of the fibers as shown in FIG. 1was affected by the cutting procedure, used to obtain cross-sections forscanning electron microscopy.

[0100] In order to be used for tissue engineering applications, fibermeshes must often be configured in a certain shape and immobilized. Thiscan be achieved by collecting the fibers in a mold, followed by bondingin a solvent-non-solvent mixture. The efficiency of this fiber bondingprocess was dependent on the solvent to non-solvent ratio of theCHCl₃/hexane mixture. Immersion of the fiber meshes in mixtures with aCHCl₃ to hexane ratio of 3:7 (v/v) did not result in stable structures.Improved bonding was obtained for devices immersed in asolvent/non-solvent mixture with a composition of 2:3. Such bonded fiberstructures were stable for several days in PBS buffer at 37° C. in ashaking bath. A bonded fiber mesh, prepared by immersion in CHCl₃/hexane1:1, remained intact for over 50 days of continuously shaking at 37° C.Solvent/non-solvent mixtures containing over 50% (v/v) CHCl₃ could notbe used, since the fibers dissolved in such mixtures.

[0101]FIG. 2 shows scanning electron micrographs of the structure of theobtained protein loaded PEG/PBT fiber meshes (cross-section (A, C) andsurface morphology (B, D) of fibers, bonded in a mixture of CHCl₃ andhexane with a volume ratio 3:7 (A, B) or 1:1 (C, D)).

[0102] As shown in FIG. 2D, confluency was observed for the structuresbonded in CHCl₃/hexane 1:1 (v/v), whereas such connections were scarcelyfound for meshes immersed in solvent/non-solvent mixtures with acomposition of 3:7 or 2:3 (FIG. 2B).

Bioactiue Agent Release from Bonded Fiber Meshes

[0103] Two different fiber meshes were selected to study bioactive agentrelease in phosphate buffered saline (PBS). The fibrous structures werebonded in a mixture of hexane and CHCl₃ of volume ratio 1:1. In order tomodulate the bioactive agent release rate, the composition of the w/oemulsion which was used to produce the loaded fibers, was varied.Previous experiments have shown that the water content in the w/oemulsion is a powerful tool to manipulate the release rate of highmolecular weight proteins. In the present, devices were prepared fromemulsions which contained a protein as the bioactive agent, in aconcentration of 1.5 or 1.75 ml of a protein solution per g of polymer,respectively.

[0104] The total protein release from the bonded fiber meshes ispresented in FIG. 3 (protein release from PEG/PBT fiber meshes, bondedin a mixture of hexane and CHCl₃ (1:1, v/v): the devices were preparedfrom emulsions which contained 1.5 (open symbols) or 1.75 ml (closedsymbols) protein solution per g of polymer (n=3; ±s.d.).). For bothdevices, a relatively large amount of protein was released during thefirst hours of incubation in the buffer. Thereafter, a slow release wasobserved, for a period longer than 10 days. A higher protein releaserate was found for the device prepared from the emulsion which containedthe highest water content.

What is claimed is:
 1. A process for preparing a polymer loaded with oneor more bioactive agents comprising the steps of: a) providing asolution of the polymer in a suitable first solvent; b) adding anaqueous solution of the bioactive agent to the polymer solution toobtain a water-in-oil emulsion; c) immersing the water-in-oil emulsionin a suitable second solvent by injecting the emulsion through a nozzleinto the second solvent; d) allowing the first solvent to migrate intothe second solvent to obtain a solid, fibrous polymer loaded with thebioactive agent.
 2. A process according to claim 1, wherein the polymeris biocompatible and biodegradable.
 3. A process according to claim 2,wherein the polymer is an amphiphilic block copolymer, comprisinghydrophilic blocks and hydrophobic blocks.
 4. A process according toclaim 3, wherein the polymer is a copolymer of a polyalkylene glycol andan aromatic ester.
 5. A process according to claim 1, wherein thebioactive agent is chosen from the group of antimicrobial agents, suchas antibacterial and anti-fungal agents, anti-viral agents, anti-tumoragents, immunogenic agents, lipids, lipopolysaccharides, hormones andgrowth factors.
 6. A process according to claim 1, wherein the bioactiveagent is chosen from the group of peptides, oligopeptides, polypeptidesand proteins.
 7. A process according to claim 1, wherein the firstsolvent is immiscible with water and miscible with the second solvent,and wherein the polymer is essentially insoluble in the second solvent.8. A process according to claim 7, wherein the first solvent has agreater solubility in the second solvent when the polymer is dissolvedin the first solvent.
 9. A process according to claim 1, wherein thewater-in-oil emulsion is immersed into the second solvent by injectingthrough a syringe or an extruder.
 10. A bioactive agent loaded polymerobtainable by the method of claim
 1. 11. A bioactive agent loadedpolymer obtainable by a process according to claim
 9. 12. A bioactiveagent loaded polymer according to claim 10 wherein said bioactive agentis a peptide, oligopeptide, polypeptide or protein.
 13. A process forbonding fibers according to claim 1 to form a fibrous mesh, wherein thefibers are collected and are bonded together by use of a suitablesolvent mixture.
 14. A fibrous mesh obtainable by a process according toclaim
 13. 15. The use of a bioactive agent loaded polymer, according toclaim 10, as a carrier for controlled drug release or as a scaffold fortissue engineering.
 16. The use of a fibrous mesh according to claim 14as a carrier for controlled drug release or as a scaffold for tissueengineering.